The invention relates to new compositions containing at least one nucleic acid and their applications in the biomedical field, particularly in gene therapy.
Up to the end of the Eighties, the use of liposomes in view of biomedical applications and, more specifically, in gene therapy did not seem very promising. The results of transfection (translation and expression of a gene of prokaryotic or eukaryotic cells by eukaryotic cells), essentially transient, were relatively mediocre. The recourse to external virus envelopes has very significantly added to the results of gene transfers in a multitude of cell lines in vitro. On the contrary, it is certain that their use in human therapy and animal therapy gives rise to problems given the possible iatrogenic dangers that they represent. Towards the end of the last decade, it appeared that the use of cationic surfactants very clearly improved the results of transfection. Since then, despite certain problems linked to the use of cationic vectors, many formulations have been proposed some of which are patented and marketed.
In general, three sorts of non-viral vectors are used:
cationic polymers,
biochemical vectors constituted of a cationic protein combined with a cell receptor,
cationic lipids associated or non-associated to liposomes.
All these vectors contain cationic molecules.
The use of such vectors is notably found described in the following publications:
J. P. Behr et al. Proc. Nat. Ac. Scienc. 86, 6982, 1989
M. Cotten and Wagner Curr. Opin. Cell Biol. 4, 705, 1993
J. Haensler and F. C. Szoka Bioconjug. Chem. 4, 372, 1993
C. P. Hodgson Bio Technology 13, 222, 1995
F. D. Ledley Hum. Gene Ther. 6, 1129, 1995
J. S. Remy et al. Proc. Nat. Ac. Scienc. 92, 1744, 1995
V. S. Trubettskoy et al. Biochem. Biophys, Acta 1131, 311, 1992
Two essential problems arise from the use of cationic vectors
1xe2x80x94these surfactants are generally cytotoxic and even if certain specific molecules have been developed in order to reduce the toxicity thereof, the problem is not entirely solved,
2xe2x80x94their application cannot be envisaged in vivo since these vectors, due to their charge, interact very strongly with proteins, for example, with those present in the serum, as well as with the cell walls. They therefore stick rapidly onto the cells neighbouring the site of injection, reducing the systemic diffusion thereof.
This is the reason why they find their preferential use in vitro for cells in culture, in the transfer of genes.
It is well known that liposomes are colloidal structures which comprise an aqueous core separated from the external medium by one or more bi-layers of phospholipid molecules. Their application in cosmetics and in pharmacology has been the subject of a large number of patents which describe many uses of these vectors since their discovery in the 1960""s.
From the point of view of applications, liposomes composed of one sole bi-layer, liposomes composed of several bi-layers often designated by the abbreviation MLV which corresponds to  less than  less than Multi-layered Vesicles greater than  greater than , have been very rapidly distinguished. The multi-layered vesicles have a size which is badly controlled and is generally far greater than a micrometer. Amongst the uni-lamellar vesicles, small vesicles, often designated as the abbreviation SUV corresponding to  less than  less than Small uni-lamellar vesicles greater than  greater than  the size of which does not exceed 100 to 300 nm, have been distinguished from large vesicles often designated by the abbreviation LUV corresponding to  less than  less than Large uni-lamellar vesicles greater than  greater than  which can attain several tens of micrometers in size.
From the fact of their significant size, the multilamellar vesicles (MLVs) are only of little use in medical applications. In fact, risks of embolism that the introduction of particles of size of greater than a micrometer into the blood circulation brings about render their use impossible by intra-venous injection. Moreover, too great a size hinders the vesicles from passing through the tissue barriers, and this enables finding them in -the blood when they are injected intramuscularly or sub-cutaneously. Finally, really efficient processes do not exist for producing MLVs in a perfectly controlled way. The major part of the applications have been developed with SUVs which fulfil the criteria of safety and control necessary for a biomedical use.
The encapsulation of nucleic acids in vesicles based on surfactants, generally lipidic surfactants, has also been described in the literature. All the vesicles described to date have in common the fact of having several layers of surfactants surrounding a liquid core. Such vesicles are described, in particular, in the patents U.S. Pat. Nos. 4,394,448 and WO-95/16437.
One of the practical problems of the use of liposomes for the vectorisation of medicaments or their use in gene therapy is the low yield due to the fact that the percentage of the starting aqueous solution effectively encapsulated rarely exceeds 30%. Moreover, the method generally followed calls for steps of evaporation of organic solvents which intervene in the method of preparation. Finally, the experimental results have revealed to be very deceiving.
In fact, the methods commonly accepted for the incorporation of lipidic vesicles, or cationic complexes which contain DNA, in the cells, pass via a mechanism of endocytosis. In the cytoplasm, the object undergoing penetration is included in an endosome which contains enzymes, in particular DNAses which are capable of destroying any intruding genetic material. The liposomes, due to their low number of lipidic membranes which surround the aqueous core, do not provide sufficient protection for resisting the destructive action of the endosomal nucleases and proteases. From this, even if they are efficient in penetrating the cytoplasm of the cell, the liposomes enable the DNA to reach the nucleus with only a low yield.
It is therefore crucial to develop synthetic vectors which are compatible with an in vivo use and this all the more so since there does not exist at present an efficient vector for molecules which can be applied in human and/or animal gene therapy. Alone, the viral envelopes show an efficiency in transfection which is compatible with a human or animal application.
The vesicles of the liposome type or paucilamellar vesicles have in common to be constituted of one or more lamellar layers surrounding an aqueous core. Apart from this type of vesicles, multi-lamellar vesicles are also known which are structurally different from the preceding ones by the fact that they have a structure known as an  less than  less than onion greater than  greater than  structure and are constituted, from their centre through to their periphery, of a succession of lamellar layers which are separated by a liquid medium. These vesicles may be obtained by a method which comprises the preparation of a liquid crystal lamellar phase and its transformation by the application of a shearing. Such a method is described in particular in the by patent WO 93/19735 originating from French patent FR-2 689 418 or WO 95/18601 introduced herein by reference.
According to French patent FR-2 689 418, this transformation may be made during a homogenous shearing step of the liquid crystal phase, and this leads to vesicles which are known as microcapsules of controlled size. However, in adjusting the formulation of the liquid crystal lamellar phase, in particular in adjusting the nature of the surfactants entering in its composition, the transformation of this liquid crystal phase into vesicles can be obtained by a simple mechanical manipulation, in particular during the mixing of the constituents.
The research conducted by the inventors have lead them to discover that the use of the technologies described above allowed developing new multi-lamellar vectors of small size, which are non-cationic, and which enable encapsulating, protecting, and delivering DNA into cells; and all this with a high encapsulation efficiency as well as with a great ease of preparation.
Another advantage is that all the molecules which enter into the preparation of the compositions of the invention are commercially available.
Another advantage of the method enabling the preparation of the compositions of the invention is that it enables using a large variety of surfactants.
According to another advantage, the invention provides a vehicle which enables, very probably due to the fact of its specific structure, protecting the nucleic acid from external attack, in particular from enzymatic attack. This point is illustrated in Example 1. This constitutes a clear advantage with respect to liposomes and to classical paucilamellar vesicles which are merely not very efficient in transfection, probably because they are not capable of protecting the DNA from the action of DNAses present in the endosome in particular. The power of the technology of the invention is partially due to this protection of the nucleic acids against nucleases. It may be supposed, without it constituting a certain explanation in the actual state of knowledge of the inventors, that the first layers of the multi-lamellar vesicle are effectively destroyed by enzymatic action in the endosome, but sufficient encapsulated DNA remains during the rupture of the endosome, so as to have a release of this DNA into the cytoplasm, which is thus capable of reaching the nucleus. Moreover, the high concentration of surfactants in the vesicles of the invention could enhance, during the erosion of the first layers, the destabilisation of the endosome by the action of the released surfactants upon the walls of the endosome.
According to another of its advantages, the invention enables, if need be, incorporating products of cationic character into the compositions, which products are already known for their importance in transfection, the other ingredients of the composition of the invention enabling masking the cationic character of this molecule, thus enabling using it for in vivo applications. Thus, the invention therefore further provides a means of increasing the efficiency or lowering the toxicity of a cationic molecule known for its use in transfection. In fact, most of the cationic compounds used are cytotoxic, and this cytotoxicity can be decreased by encapsulating these molecules, and this constitutes a further advantage of the invention.
Other advantages appear from the description and the Examples which follow, wherefrom it may be seen that this type of vector enables significantly overcoming all the problems set forth above and enables offering, for the first time, a vehicle, in the form of vesicles based on surfactants, which may be used in vivo.
The invention relates, according to one of its essential characteristics, to a composition which contains at least one nucleic acid at least one part of which is found included inside multilamellar vesicles, characterised in that said vesicles are constituted of bi-layers comprising at least one surfactant agent, said bi-layers being concentric and giving an onion structure to said vesicles.
 less than  less than Onion greater than  greater than  structure is understood as meaning, as set forth above, a multi-lamellar structure in which the vesicles of substantially spherical shape are constituted of a succession of concentric bi-layers, this being from the centre to the periphery of the vesicles, from which the name of onion structure is used, by analogy, for qualifying such structures.
Such structures are advantageously obtained by incorporating at least one nucleic acid in a liquid crystal lamellar phase which comprises at least one surfactant agent, and then transforming this lamellar liquid crystal phase into a dense phase of small-sized multi-lamellar vesicles.
Thus, according to another essential characteristic of the invention, the invention relates to a method of preparation of a composition which contains at least one nucleic acid as defined above, according to which method, a lamellar liquid crystal phase is prepared which incorporates said nucleic acid, and the rearrangement of said liquid crystal phase into multi-lamellar vesicles is caused by the application of a shearing.
This shearing may be a homogenous shearing, and this has the advantage of leading to vesicles of perfectly homogenous size. However, a simple mechanical stirring may prove to be sufficient to lead to the formation of the multi-lamellar vesicles of the invention.
According to yet another characteristic, the invention relates to the products which are obtainable by this method.
According to French patent FR-2 689 418, this transformation may be made during a homogenous step of shearing of the liquid crystal phase, and this leads to vesicles or microcapsules of controlled size. However, in adjusting the formulation of the liquid crystal lamellar phase, in particular in adjusting the nature of the surfactants entering in its composition, the transformation of this liquid crystal phase into vesicles can be obtained by simple mechanical manipulation, in particular during the mixing of the constituents.
The formulation advantageously includes a mixture of surfactant molecules. At least two different surfactants are generally used which have different hydrophilic lipophilic balances, and this enables continuously regulating the properties of the bi-layers, and thus enables controlling the appearance of instability which governs the formation of the multi-lamellar vesicles.
According to an advantageous variant, the vesicles which constitute the compositions of the present invention are of dimensions which are less than 1 xcexcm, preferably between 0.1 and 1 xcexcm.
According to another advantageous variant of the invention, the surfactants which constitute the bi-layers of the vesicles are non-cationic surfactants, and this, as has been seen previously, has a great advantage over the compositions of the prior art which make use of cationic molecules.
According to an advantageous variant, the membranes of the vesicles contained in the compositions of the invention advantageously contain at least one surfactant agent selected from the group consisting of:
hydrogenated or non-hydrogenated phospholipids,
saturated or mono- or polyunsaturated, linear or branched C6 to C18 fatty acids, in the form of an acid or an alkali metal salt, an alkaline earth metal salt, or an amine salt,
ethoxylated or non-ethoxylated esters of the same fatty acids and
of sucrose,
of sorbitan
of mannitol,
of glycerol or of polyglycerol,
of glycol,
mono-, di- or triglycerides or mixtures of glycerides of the same fatty acids,
saturated or mono- or polyunsaturated, linear or branched, ethoxylated or non-ethoxylated C6 to C18 fatty alcohols,
ethoxylated or non-ethoxylated ethers of the same fatty alcohols and
of sucrose,
of sorbitan,
of mannitol,
of glycerol or of polyglycerol,
of glycol,
hydrogenated or non hydrogenated polyethoxylated vegetable oils,
block polymers of polyoxyethylene and polyoxypropylene (poloxamers),
polyethylene glycol hydroxystearate,
sterol-skeleton alcohols such as cholesterol, sistosterol.
According to an advantageous variant, the surfactants entering in the composition of the bi-layers of the vesicles are constituted of non-cationic surfactants.
The surfactants selected, in particular the surfactants cited above, are advantageously selected from the category of surfactants which are allowed by the legislation for pharmaceutical use as a function of the route of administration.
Two surfactants having relatively different properties will advantageously be chosen from the surfactants above, in particular ones having a different hydrophilic lipophilic balance (HLB). The first surfactant will advantageously have a hydrophilic lipophilic balance between 1 and 6, while the second surfactant will have a hydrophilic lipophilic balance between 3 and 15.
As has been seen above, a better control of the size of the multi-layer vesicles can be obtained in proceeding according to French patent FR 2 689 418.
The preparation obtained after the transformation of the liquid crystal lamellar phase into multi-lamellar vesicles may then be diluted, in particular with an aqueous solvent in order to thus obtain an aqueous suspension of vesicles.
As set forth above, one of the drawbacks of the nucleic acid vectors known to date, in particular liposome-type vectors, is to only rarely enable exceeding encapsulation yields of 30%. The method used for the preparation of the vesicles according to the invention enables, itself, reaching yields of encapsulation which can be in the region of 100%. However, such an encapsulation yield, due to the fact of the high activity obtained by virtue of the particular structure of the vesicles of the invention does not always prove necessary.
Thus, the encapsulation yield of the nucleic acid(s) in the compositions of the invention is advantageously at least 10%, preferably at least 40% but can also be between 60 and 100%, and this represents a further advantage with respect to the technologies of the prior art.
The compositions of the invention which contain vesicles which include at least one nucleic acid may be used for in vitro applications, in particular for transforming a cell line, in particular for immortalising or modifying the expression of one or more genes therefrom.
The invention provides a means of vectorization of nucleic acids, it being possible for this vectorization to be carried out in vivo as well as in vitro.
These vesicles can also be used for the preparation of pharmaceutical compositions which can be used in gene therapy.
DNA or a nucleotide sequence of DNA can be cited as examples of a nucleic acid which can be contained in the vesicles of the invention.
The vesicles can also include a particular gene or a sequence of this gene, more particularly a sequence which encodes a given protein.
The compositions of the invention can also include RNA or a nucleotide sequence of RNA in the vesicles.
According to another variant, an oligonucleotide may also be included in the vesicles, it being possible for this oligonucleotide to be of sense and/or anti-sense type.
The vesicles of the invention are advantageously less than 1 xcexcm in size, preferably between 0.1 and 1 xcexcm. According to the method of the patent FR-2 689 418 or WO/FR93/19735, it will be possible for this size to be controlled and to be limited by the application of a homogenous shearing of the liquid crystal phase.
According to particularly interesting variants of the invention, it will be possible for various products to be co-encapsulated with the nucleic acid with the view, in particular, to improving the conditions of action of this nucleic acid, or to bring about supplementary functionalities to that of the product encapsulated.
It will be possible for such compositions to be produced by the method described above, in incorporating the product to be co-encapsulated in combination with the nucleic acid in the liquid crystal phase.
Amongst the products that may be advantageously co-encapsulated with the nucleic acids, products of the  less than  less than condensation agent greater than  greater than  type will be cited in particular which form a sort of complex with the nucleic acid which stimulates the  less than  less than folding greater than  greater than  of the nucleic acid chain, and this enhances its stability in the presence of nucleases.
The histones, which are proteins, and which exist naturally in the nucleus for condensing DNA, will be cited as a preferred example of condensation agents. Histones have the particularity of remaining perfectly bound to the DNA and thus are susceptible of playing an important role in many genomic reactions. Histones known as nucleosonic histones, which are responsible for the folding of DNA into nucleosomes, more specifically, histones H2A, H2B, H3 and H4, and the histone H1 which does not directly participate in the replication of DNA into nucleosomes, but to the stacking to the latter, will be cited amongst the histones which will advantageously be encapsulated in combination with at least one nucleic acid, in particular with DNA. The use of a mixture of histones Hi, H2A, H2B, H3 and H4 (originating from the thymus of the calf) used with a ratio of 1 xcexcm of total proteins per xcexcg of DNA has thus enabled condensing 50 xcexcg of DNA, with a maximum encapsulation yield, into the vesicles of the invention.
According to other variants of the invention, various enzymes, in particular integration enzymes, recombination enzymes or enzymes intended to optimise the replication of the nucleic acid encapsulated, such as the toposomerases or helicases, will advantageously be co-encapsulated into the multi-lamellar vesicle of the invention.
Thus, according to a variant of the invention, integration enzymes may be co-encapsulated in the multi-lamellar vesicle, the function of which enzymes is to enable the gene introduced into the cell to express itself in a host cell, but, above all, to integrate itself with the genome to give daughter cell lines which keep the function brought about the gene introduced. To date, only the use of retroviruses as DNA vector enables a stable and efficient transfer of genes, via the integration of the viral genome into the host cell. However, all the problems linked to the use of retroviruses in the biomedical field may not be brushed aside. Thus, the power of the technology of the invention is to be able to co-encapsulate DNA with proteins, in particular enzymes which enable this integration, which enzymes are designated by  less than  less than integration enzymes greater than  greater than . Thus, DNA and the tools necessary for its incorporation may thus be brought about in a same vector.
It is thus possible to stimulate this genomic integration by the addition of recombination enzymes, specific of the site, which allow introducing, even eliminating, given nucleotide sequences. This form of recombination is called  less than  less than site specific recombination greater than  greater than  since the recombination enzyme called integrase recognises nucleotide sequences which can be introduced into a DNA vector which is susceptible to recombination with the DNA from the host cell. This recombination enzyme approaches specific sites, initiates the reaction of cutting, and of soldering of the DNA.
Other enzymatic tools may also be integrated in the vector. The DNA is replicated in eukaryotic organisms as chromatin in which the DNA is combined strongly with the histones, proteins which may also be used according to the present invention for optimising the quantity of DNA encapsulated. If this condensed structure of stacked nucleosomes acts as a barrier which would stop for example the enzymes responsible for replication, the combination of the DNA condensed with the enzymes such as the topoisomerases or the helicases enables solving the eventual problems of winding of the helix or the opening of same.
As has been seen before, one of the advantages of the compositions of the invention is that they provide a nucleic acid vehicle which does not contain any molecule of cationic character.
However, according to a variant of the invention, it is possible to include certain cationic adjuvants, the presence of the multi-lamellar vesicles of the invention enabling masking their toxicity or increasing their activity. It seems that the effect of certain cationic adjuvants is to enable electrostatically fixing the vesicles onto the cell walls, and this explains the increase in the efficiency of the vectors. Furthermore, it appears that the fact of encapsulating a part of the cationic molecules enables lowering their cytotoxicity, the encapsulation decreases the percentage of cationic molecules which are susceptible of entering in contact with the neighbouring cells.
The encapsulation also enables reducing the concentration of cationic molecule used, by virtue of the synergistic effect which increases the efficiency thereof.
Thence, it is possible to co-encapsulate a cationic adjuvant into the vesicles according to the invention in preventing the drawbacks known according to prior art during the use of such products.
It will also be possible for the vesicles comprising the nucleic acid, according to the particularly interesting variants of the invention, to be modified on the surface with the view to be better recognised by the target sought after. According to this variant, molecules, in general proteins, will be attached to the vesicles, and this will enable them to be recognised specifically by certain targets in the organism, and therefore enable them to increase the selectivity of the transfection on the one hand, and on the other hand, the efficiency thereof. Several methods may be used, in particular recourse to monoclonal antibodies. In every case it is necessary to fix the targeting system onto the surface of the vesicle, in order that its recognition efficiency be maximal. This fixation may be carried out by physical methods (adsorption) or even chemical methods. In both cases, the ease of obtaining and manipulating vesicles of the invention are the major advantages for the success of these graftings of functions onto the surface of the vectors. One of the important applications in the biomedical field is the use of vesicles which have on their surface monoclonal antibodies or Fab fragments, which are specific to receptors of the cell surface implied, for example, in the recognition of viral particles (herpes virus . . . ) and which act as transductor of the cell response.
The invention, according to other essential characteristics, relates to pharmaceutical compositions which contain vesicles as defined above which contain various nucleic acids, in suspension in a pharmaceutically acceptable vehicle.
The invention also relates to the methods of therapeutic treatment which use these compositions.
According to the biological or pharmaceutical activity sought after, various types of nucleic acids may be encapsulated.
Thus, it will be possible for the encapsulation of a complete gene to be carried out with the view to providing a pharmaceutical composition intended to provide a deficient gene or an absent gene. As an example, it will be possible for such a use to be made in a treatment of mucoviscidosis.
The encapsulation of nucleic constructions such as oligonucleotides intended to induce an over-expression of or a reduction in the activity of a specific gene will be able to be used in the applications in cancerology or in virology.
These vectors can also be used in models of vaccination from DNA which encodes an antigenic protein.
RNA can also be vectorized since the vectorization of a complementary nucleotide sequence hybridising to transcript inhibits the reading of the mRNA by the ribosomes and prevents the translation thereof into an active protein.
The invention also concerns the use of compositions defined above as an agent intended to transform a cell line.
The invention therefore relates to a method of in vitro transformation of a cell line, which consists in treating said line with a composition as described above.
More specifically, in this case, a gene is brought up to the level of the nucleus of the cell so that it be intranuclear and/or intrachromosomic in order to modify its proper expression of it and/or the expression of other genes which encode one ore more proteins, or to immortalise a line by the incorporation of a viral gene and/or a viral oncogene.
According to a further advantage of the technology of the invention, the invention provides vesicles which enable carrying out a transfection in the presence of serum, and this is impossible to carry out with vesicles of the prior art. This constitutes a considerable progress which enables envisaging for the first time the use of surfactant-based vesicles in vivo.
This point was verified by in vitro transfection tests on cell lines, and on primary cultures of differentiated human cells. It has been possible for results of transfection, which are as good in the presence of foetal calf serum as in the absence of foetal calf serum, to be obtained when other artificial vectors known at the present time (cationic lipids) do not function in such a medium. The latter result is fundamental because it is a sine qua non condition of in vivo use.
The first in vivo transfection results are very encouraging. These results have shown that the transfer of genes could take place in a tumour site and in systemic injection. The intravenous injection, in the animal, of vesicles according to the invention encapsulating the xcex2-galactosidase gene enables inducing a galactosidase activity in various organs (the heart, the lungs and the liver). This point is very encouraging for the use in therapeutic goals of the procedure.
Thus, the invention also relates to the use of multi-lamellar vesicles of onion structure for the preparation of a pharmaceutical composition intended to correct or to alleviate a deficient or altered cell function or to bring about a new function to a cell, notably in the field of cancerology, of virology or in the correction of bone defects.
Thus, the applications of this vector touch several biomedical research fields, in particular cancerology. In this field, the first results have been obtained in mice bearing tumours. Injections of vesicles which encapsulate a reporter gene have been carried out and a gene activity has been identified at the level of the tumour. It therefore becomes possible to envisage the use of such vectors which bear genes which encode antimitotic factors, or implicated in apoptosis phenomena (cell death) in order to study the effect of this transfer of genes upon the regression of the tumour.
Moreover, another advantage of this vector is to also function in vivo at the level of a given implantation site. Another field of application of these vectors relates to the actual methods of bone substitution. The implantation, in a bone fault, of vesicles which bear the gene which encodes the osteoinducing proteins known by the term  less than  less than BMPs greater than  greater than  which corresponds to  less than  less than Bone Morphogenetic Proteins  greater than  greater than , would stimulate osteogenesis. If the future of these osteoinducing factors in the orthopaedic field is very promising, at the present time, the major problem of the vectorization, of the regulation of the delivery system at the site of the implantation without observing any systemic effect, does nevertheless remain to be solved. The present invention provides for the first time an acceptable vector. Furthermore, the systemic injections of vesicles bearing the xcex2-galactosidase gene, and the first biodistribution experiments have shown a genic activity in various organs. The use of such a method combined with a targeting, as described above, offers great therapeutic perspectives.